During intracellular transport, proteins destined for the plasma membrane, secretory vesicles, and lysosomes must be sorted from one another within the Golgi complex, and sent to their appropriate addresses. The long term goal of this research is to elucidate the molecular mechanisms by which proteins are targeted to specific and distinct intracellular destinations. The targeting of proteins to lysosomes is presently the best understood sorting process. Soluble lysosomal enzymes acquire a mannose 6-phosphate (man6P) tag; intracellular receptors recognize man6P-containing proteins, and somehow facilitate their transport to lysosomes. The specific aims of this proposal are: 1. To study protein sorting in the trans Golgi network (TGN), clathrin coated vesicles carrying man6P receptors will be isolated by immunoadsorption. Their content of a lysosomal membrane glycoprotein will then be analyzed to determine if these two proteins are transported together from the TGN to pre-lysosomes. 2. To study protein sorting in pre-lysosomes, the potential recycling of a lysosomal membrane glycoprotein from pre-lysosomes to the TGN will be investigated. The ability of a soluble lysosomal enzyme to be transported to the TGN will also be examined. 3. To study the molecular basic of vesicular transport from pre-lysosomes to the TGN, an analysis of the cytosolic factors that facilitate this process will be initiated using a cell-free system developed in this laboratory, Purification of an N-ethylmaleimide-sensitive factor will attempted. Experiments will be carried out to examine the potential role of clathrin and a transport factor termed "NSF" in this transport process. Finally, an anti-peptide antiserum will be generated to identify a family of GTP binding proteins that facilitates intracellular transport. It is hoped that these cell biological and biochemical experiments will further our understanding of the molecular mechanisms by which proteins are transported between membrane-bound compartments within the cytoplasm of eukaryotic cells.